Single-cell RNA profiling of Plasmodium vivax-infected hepatocytes reveals parasite- and host- specific transcriptomic signatures and therapeutic targets

Abstract

The resilience of Plasmodium vivax, the most widely-distributed malaria-causing parasite in humans, is attributed to its ability to produce dormant liver forms known as hypnozoites, which can activate weeks, months, or even years after an initial mosquito bite. The factors underlying hypnozoite formation and activation are poorly understood, as is the parasite’s influence on the host hepatocyte. Here, we shed light on transcriptome-wide signatures of both the parasite and the infected host cell by sequencing over 1,000 P. vivax-infected hepatocytes at single-cell resolution. We distinguish between replicating schizonts and hypnozoites at the transcriptional level, identifying key differences in transcripts encoding for RNA-binding proteins associated with cell fate. In infected hepatocytes, we show that genes associated with energy metabolism and antioxidant stress response are upregulated, and those involved in the host immune response downregulated, suggesting both schizonts and hypnozoites alter the host intracellular environment. The transcriptional markers in schizonts, hypnozoites, and infected hepatocytes revealed here pinpoint potential factors underlying dormancy and can inform therapeutic targets against P. vivax liver-stage infection.

Transcriptional profiling of hepatocytes infected with the replicative form of the malaria parasite Plasmodium cynomolgi

“…1 ). Moreover, the study by Ruberto et al [ 27 ] reported opposite modulations of host pathways (e.g., upregulations of host genes associated with translation and oxidative phosphorylation) in hepatocytes infected with P. vivax , which is in apparent disagreement with our results, as well as with results from others [ 23 , 26 ]. Thus, beyond the impact of malaria- and host-species specific variations, current results suggest that the hepatocyte response to infection is highly dynamic or influenced by technical aspects (e.g., the source of human hepatocytes).…”

“…Recent studies reported the host transcriptional profile of human hepatocytes infected with P. vivax [ 26 , 27 ]. Interestingly, Mancio-Silva et al [ 26 ] found that the late phase of the P. vivax liver stage is associated with the downregulation of liver-specific functions as well as translation, as observed for P. cynomolgi - and P. berghei -infected hepatocytes (Figs.…”

A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways inPlasmodium vivaxHypnozoites

“…The hit MS-0735, an analog of our previously reported hypnozonticidal hit MMV018983 ( 13 ), is a ribonucleotide-reductase (RNR) inhibitor used as an antiviral. Needed for producing deoxyribonucleosides for DNA synthesis, RNR is a peculiar mechanism for nonreplicating hypnozoites, however, it has been reported that RNR is critical for DNA damage repair ( 38 ) and is expressed in P. vivax liver schizonts and hypnozoites ( 19 ). Ongoing efforts to make available disruptive methods for studying P. vivax , including genetic manipulation and in vivo models for relapse, will enable further validation of these leads and a more comprehensive understanding of the mechanisms of hypnozoite quiescence.…”

“…Similarly, the cancer drug poziotinib inhibits HER2, a tyrosine protein kinase associated with the downregulation of apoptosis and metastasis (37). As we recently reported that host apoptotic pathways are downregulated in P. vivax-infected hepatocytes, poziotinib could act by increasing apoptotic pathways in infected host cells (19). The hit MS-0735, an analog of our previously reported hypnozonticidal hit MMV018983 (13), is a ribonucleotide-reductase (RNR) inhibitor used as an antiviral.…”

“…We next sought to confirm the presence of cytosine methylation in the P. vivax and P. cynomolgi genomes using mass spectrometry and bisulfite sequencing. Initially, we assessed if these methodologies were plausible on P. vivax liver stages, but concluded that, without an available single live-cell approach, sequencing coverage of the parasite’s genome would be overwhelmed by the genomic material from the host cell and neighboring uninfected hepatocytes following the harvesting of liver stage cultures ( 19 ). We therefore collected sufficient genomic material from P. vivax and P. cynomolgi sporozoites to analyze the nucleoside mixture arising from the enzymatic digestion of genomic DNA by liquid chromatography-tandem mass spectrometry as well as for detection of DNA methyltransferase (DNMT) activity using a commercial in vitro DNA methylation assay ( 17 ).…”